PROJECT SUMMARY Opiates targeting the mu opioid receptor (MOR) are broadly effective pain relieving drugs, but opiate abuse is at record levels in the United States. In spite of the magnitude of opiate abuse and dependence, our understanding of how MOR agonist exposure causes reward and addiction is limited. The overarching objective of this proposal is to investigate the circuits that contribute to MOR mediated reward. We will focus on the connections between the ventral tegmental area (VTA) which is required for MOR mediated reward (Olmstead and Franklin, 1997; Zhang et al., 2009), and the lateral habenula (LHb), which is activated by a range of aversive states including pain. Although dopamine in the ventral striatum is required for morphine reward in opiate dependent animals, it is not required in nave animals (Bechara et al., 1992; Nader and van der Kooy, 1997). Therefore to understand the transition to opioid dependence and abuse it is essential to determine the non-dopaminergic VTA output circuit that mediates MOR reward. The VTA projection to the LHb is non-dopaminergic (Root et al., 2014) and is a potential candidate for MOR reward. Since LHb neurons are activated by painful stimuli (Benabid and Jeaugey, 1989; Zhang et al., 2013) and stimulating VTA glutamate projections to LHb produces aversion (Root et al., 2014), MOR inhibition of VTA glutamate projections to the LHb should be rewarding. Our preliminary data show MOR induced inhibition of this VTA projection. Our preliminary data also show direct MOR inhibitions of LHb neurons that project to the GABAergic RMTg, which signals reward by disinhibiting VTA dopamine neurons. We will fully characterize the MOR effects in the VTA- LHb circuits, including studying the mechanisms of MOR action at each synaptic site and determining the projection target(s) of the LHb neurons hyperpolarized by MOR activation. In conjunction with the electrophysiology, we will perform single cell nCounter detection of 32 target mRNAs in recorded neurons to enable full characterization of the neurotransmitters released (or co-released) by the recorded neurons and to identify novel receptor targets. We will use behavioral pharmacology and optogenetics to determine the contribution of these circuits to MOR reward in both nave animals and in the spared nerve injury model of neuropathic pain, since pain activates the LHb and leads to changes MOR function in various brain regions including the VTA (e.g. Hipolito et al., 2015). Given that both reward due to relief of chronic pain and opioid reward in opioid dependent animals require dopamine, we will probe the state dependent changes in MOR function in the VTA-LHb circuits using slice electrophysiology. Through comparing mRNA expression in the individual neurons in these circuits in nave, chronic pain, and morphine dependent animals, we will be able to determine the molecular and synaptic changes that underlie the transition to opioid dependence and identify potential targets for future drug development. This work will enable development of more effective therapeutic agents for pain that can circumvent the problems of opioid tolerance, dependence and abuse.